The present invention, in some embodiments thereof, relates to plant viral expression vectors and, more particularly, but not exclusively, to the use of same for generating genotypic variations in plant genomes.
Genetic modification and improvement of crop plants as well as protection of new varieties is fundamental for modern agriculture. During the past several years an enormous amount of data was obtained from the various large genome-sequencing projects allowing significant progress in agriculture transgenic technologies. Such technologies, including gene expression, gene modification, site-specific gene mutagenesis and gene targeting of plant genome sequences, allow development of basic plant research models and can be directly used for genetic improvement and protection of agronomically important plant species.
Foreign DNA molecules (e.g. T-DNA) delivered by Agrobacterium are integrated in the plant's genome into natural double strand breaks (DSBs) which may be generated by rare-cutting restriction enzymes. These DSBs are recognized and repaired by plant non-homologous end joining (NHEJ) proteins and results in the frequent integration of the foreign DNA into these random sites [Salomon et al. EMBO J (1998) 17: 6086-6095; Tzfira et al. Plant Physiol (2003) 133: 1011-1023, Tzfira et al. Trends Genet (2004) 20: 375-383]. The DSBs may also result in enhanced homologous recombination (HR)-based gene targeting in plant cells [Puchta et al. Proc Natl Acad Sci USA (1996) 93: 5055-5060].
Recent developments in the field of zinc finger nucleases (ZFNs) as novel tools for genome modifications offer new prospects for site-specific induction of DSBs in plant genomes and for the development of NHEJ-based methods for gene targeting in plant species and plant protection. ZFNs are synthetic restriction enzymes which can be specifically designed to bind and cleave virtually any long stretch of dsDNA sequences (see FIG. 1). ZFNs were shown suitable for site-specific genomic DSB induction in plant species using non-viral vectors [Lloyd et al. Proc. Natl. Acad. Sci. U.S. A. (2005) 102: 2232-2237; Tovkach et al. The Plant Journal (2009) 57, 747-757]. Similar effects were shown on human [Moehle et al. Proc Natl Acad Sci USA (2007) 104: 3055-3060] and insect genomes [Beumer et al. Genetics (2006) 172: 2391-2403].
The use of plant viruses as vehicles to introduce and express nonviral genes in plants is well documented [e.g. Donson et al., Proc Natl Acad Sci USA. (1991) 88: 7204-8; Chapman et al. Plant J. (1992) 2: 549-57; Dolja et al., Virology (1998) 252: 269-74]. Infection of plants with modified viruses is simpler and quicker than the regeneration of stably transformed plants (as discussed above) since plant viruses are often small in size (between 3000 and 10,000 nucleotides), are easy to manipulate, have the inherent ability to enter the plant cell, lead to the immediate expression of the heterologous gene and will multiply to produce a high copy number of the gene of interest. Viral vectors have been engineered for delivery of genetic material and expression of recombinant proteins in plants [e.g., Pogue, Annu. Rev. Phytopathol. (2002) 40: 45-74; Gleba, et al., Curr. Opin. Plant Biol. (2004) 7: 182-188; Dolja et al., Proc. Natl. Acad. Sci. USA (1992) 89: 10208-10212; U.S. Pat. No. 5,316,931 and U.S. Pat. No. 5,811,653 for RNA virus vectors]. Viral expression systems are considered transient expression systems as the viral vectors are not integrated into the genome of the host, however, depending on which virus is used, virus multiplication and gene expression can persist for long periods (up to several weeks or months).
To date the use of viral vectors for introducing DSBs in plant genomes was not demonstrated or suggested.